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SMITH, KUPKE, LOEFFLER, BENITEZ, AHRNE, GIESE 



e.g., near 072 m/x. With aqueous buffer extracts the shift was very 

 rapid; this may account for the short wavelength position found 

 by the Russian workers. It is very doubtful, therefore, whether a 

 shift in the absorption maximum of native chlorophyll results from 

 chlorophyll accumulation during the greening process. 



Although an active form of the protochlorophyll holochrome can be 

 extracted from etiolated leaves by glycerine, such extracts are diffi- 

 cult to work with for the purpose of isolating the holochrome. Be- 

 cause of the technical difficulties involved in the use of glycerine. 



600 



900 



700 800 



WAVELENGTH 



Fig. .3. The transformation of protochlorophyll holochrome to chlorophyll-a 

 holochrome in phosphate buffer extracts of barley and bean leaves and of a mix- 

 ture of the two. The buffer solution, pH 7.08, contained 0.02 M phosphate and 0.01 

 M potassium chloride. 



we have undertaken the isolation of the holochrome from aqueous 

 buffer extracts of the dark-grown leaves. Krasnovskii and Koso- 

 butskaya (6) were the first to obtain active extracts of the holochrome 

 in an aqueous medium. They extracted etiolated bean leaves with 

 phosphate buffer of pH 7. We have confirmed their results, but we 

 have found that, of the three sources which we examined (barley 

 leaves, bean leaves, and squash cotyledons) , only bean leaves yield 

 an active protochlorophyll holochrome in aqueous buffer solutions 

 (Fig. 3). Barley-leaf extracts in phosphate buffer, pH 7.08, contain 

 practically no protochlorophyll and show no transformation. They 



